Process for suppressing foot-and-mouth disease virus

ABSTRACT

Certain water-soluble copolymers prepared from divinyl ether and maleic anhydride have been found to suppress foot-and-mouth disease virus growth in cloven-footed animals.

United States Patent Continuation-impart of application Ser. No.716,257, Mar. 6, ,i 968, now abandoned which is a continuation-in-partof application Ser. No. 577,675, Sept. 7, [966, now abandoned. Thisapplication July 10, 1970, Ser. No. 54,021

[54] PROCESS FOR SUPPRESSING FOOT-AND-MOUTH DISEASE VIRUS 2 Claims, NoDrawings [52] US. Cl.. 424/78 [5 l] int. Cl. A6lk 27/00 [50] Field ofSearch 424/78;

[56] References Cited UNlTED STATES PATENTS 3,224,943 12/1965 EspyPrimary Examiner-Jerome D. Goldberg An0rney--.lohn W. Whitson ABSTRACT:Certain water-soluble copoiymers prepared from divinyl ether and maleicanhydride have been found to suppress foot-and-mouth disease virusgrowth in eleven;

footed animals.

PROCESS FOR SUPPRESSING FOOT-AND-MOUTH DISEASE VIRUS This application isa continuation-in-part of my copending application Ser. No. 716,257,filed Mar. 6, 1968, now abandoned which is in turn acontinuation-in-part of my application Ser. No. 577,675, filed Sept. 7,1966, now abandoned.

This invention relates to a method of suppressing foot-andmouth diseasevirus growth in cloven-footed animals. More specifically, this inventionrelates to a method of inhibiting or retarding foot-and-mouth diseasevirus growth and preventing its propagation by inactivation of the virusand/or increasing host resistance to the virus infection.

Foot-and-mouth disease is caused by a filterable virus classified intoseveral different types. Cloven-footed animals are the natural hosts offoot-and-mouth disease virus. Cattle, oxen pigs, sheep, goats, wildruminants, hedgehogs, and even some rats are susceptible to naturalinfection. Guinea pigs, hamsters, rabbits, mice, cats and dogs may beinfected experimentally, but do not usually develop the diseasenaturally. Animals usually are infected with the virus by direct contactwith another infected animal. However, indirect contact is common due tothe fact that the virus can survive for long periods outside a livinganimal. The incubation period after natural infection is usually between3 and 8 days, but can be shorter or may extend to 14 days or longer. Thenormal procedure for controlling an outbreak of foot-and-mouth diseaseis the slaughter and destruction of infected animals along withrestrictions on the movements of people, animals and materials.Obviously an outbreak of the disease can cause severe economic andsocial problems. Vaccines have been used in some countries with varyingsuccess. However, vaccination with one type of vaccine may not beeffective in preventing infection with other virus types or sub types.

It has now surprisingly been discovered that certain water- 'solublecopolymers prepared from divinyl ether and maleic anhydride in moleratios of 1:2 and their physiologically tolerated salts are effective insuppressing foot-and-mouth disease virus. These copolymers arecharacterized by having an RSV of from about 0.04 to about 1.8 and ashaving the following recurring unit:

It will be obvious to those skilled in the art that these copolymerswill hydrolyze on contact with water to produce the free acid.

The term Reduced Specific Viscosity (RSV), which is a function ofmolecular weight, is used herein to designate the specific viscositymeasured at a temperature of C. on a 0.1 percent solution of thecopolymer in a one molar aqueous solution of sodium hydroxide.

Unlike some antiviral agents, the copolymers used in this invention arerelatively nontoxic. For example, when injected intraperitoneally inmice they were found to have an LD of greater than 800 mg./kg. By theterm LD is meant lethal dose to 50 percent of the animals being tested.

Most of the chemotherapeutic agents currently in use in the treatment ofvirus infections exert their activity through direct physicalcombination with the virus or by modifying the ability of the virus toattach or replicate or to be released from within the infected cell.

The exceptionally high activity of these copolymers as antiviral agentsis multiple in that they are active both in vitro and in vivo. Theypossess in vitro viral neutralization by direct physical combinationwith the virus. On the other hand, they exhibit surprising in vivoactivity in reducing the quantity of virus present in the injectedtissue following virus infection.

They also increase the resistance of healthy animals to footand-mouthdisease virus infection on pretreatment by stimulating thereticuloendothelial system to induce the production of interferon.Interferon is a protein produced by animal and human cells whichpossesses antiviral activity against a wide spectrum of viral pathogens.

The copolymers can be employed in aqueous solution or dissolved inphysiological sterile saline solution. In addition, variouspharmaceutical preparations can be compounded which contain the activesubstance along with liquid or solid diluents. Solid preparations forextemperaneous dilution can be formulated employing various bufferingagents as well as local anesthetics and other medicinal agents such asantibiotics, hypnotics, analgesics, etc., and inorganic salts to afforddesirable pharmacological properties to the composition. Administrationwill be by one of the conventional intramuscular, subcutaneous,intravenous or intraperitoneal routes.

Divinyl ether-maleic anhydride copolymers can be used to treat clovenfooted animals which are actually infected with foot-and-mouth diseasevirus or to immunize animals which may be exposed to the virus. Forexample, if the disease breaks out in a herd of cattle the owner wouldadminister the copolymer to those animals exhibiting symptoms of thedisease as well as those which look healthy and owners of neighboringherds would immunize their animals because they might be exposed to thevirus. Thus the copolymer acts not only as a medication, but as aprophylatic. In any case, dosages in the order of 0.5 to 300 mg./kg.daily of the copolymers are highly effective in suppressingfoot-and-mouth disease virus in cloven-footed animals and in increasingresistance to the disease in healthy animals by inducing the productionof interferon. The specific dosage will depend upon the route ofadministration and duration of treatment. Since the active copolymersare stable and widely compatible, they can be administered in solutionor suspension in a variety of pharmacological acceptible vehiclesincluding water, propylene glycol, diethylcarbonate, glycerol, or oilssuch as peanut oil, sesame oil, olive oil, etc.

As stated above, the physiologically tolerated salts of the copolymerscan be used in the process of this invention. Exemplary salts are thewater-soluble salts of alkali metals as for example sodium, potassium,etc.; ammonium salts, salts of such amines as methylamine,dimethylamine, ethylamine, diethylamine, butylamine, aniline,methoxyamine, piperidine, morpholine, etc.; mixed salts containingammonia and a primary or secondary amine, etc.

The copolymers used in the process of this invention can be produced bycopolymerizing divinyl ether and maleic anhydride in an aromatic diluentat a mole ratio of about l:2 using a free radical initiator (e.g.benzoyl peroxide, azo bis- (isobutyronitrile), etc.) The use of chaintransfer agents (i.e. CCL, BrCCl etc.) in the copolymerization isoptional.

The following example shows the preparation of a typical divinylether-maleic anhydride copolymer.

A polymerization vessel was charged with 197.2 parts of maleicanhydride, 704 parts of benzene and 1276 parts of carbon tetrachloride.After dissolution of the maleic anhydride, the solution was sparged withnitrogen and 70.2 parts of distilled divinyl ether was added withagitation. Then with vigorous agitation there was added 1.45 parts ofbenzoyl peroxide dissolved in benzene. Within 20 seconds,copolymerization started and the solution became cloudy and thengelatinous. After about 3%hours, the swollen polymer was removed andrepeatedly extracted with benzene and then dried under vacuum. Theresulting divinyl ether-maleic anhydride copolymer had an RSV of 0.34(molecular weight of approximately 36,000) and represented a conversionof 89 percent. The divinyl ether and maleic anhydride were present inthe copolymer in the molar ratio of l :2.

It will be understood that by conducting the copolymerization undervarious reaction conditions in the presence of different free radicalinitiators and in the presence or absence of chain transfer agents,copolymers of various molecular weights can be prepared.

The following examples are presented to illustrate th process of thisinvention.

EXAMPLE I This example illustrates the induced production of interferonby treating Swiss white mice with a divinyl ether-maleic anhydridecopolymer containing divinyl ether and maleic anhydride in 1:2 moleratio and having an RSV of 0.34 (molecular weight of approximately36,000). Six groups containing five mice each were all injectedintraperitoneally with I25 mg./kg. body weight of the copolymer insterile saline solution. The groups of mice were sacrificed as follows:the first group, immediately; the second after 12 hours; the third after24 hours, the fourth after 48 hours, the fifth after 72 hours; and thesixth after 144 hours. The pooled mouse serum from each group was testedto determine the units of interferon produced according to the procedureof Kleinschmidt et al. P.N.A.S. 52: 74l, 1964. The results of thesetests are tabulated below along with results of control groups of fivemice each injected with sterile saline solution alone:

Units of Interferon Present This example illustrates-the production ofinterferon using divinyl ether-maleic anhydride copolymers of varyingmolecular weight. Four groups of Swiss white mice containing five miceeach received intraperitoneal injections of I25 mg./kg. body weight ofthe copolymer in sterile saline solution with each group receiving adifferent molecular weight copolymer. All of the mice were sacrificed 24hours after treatment and the pooled mouse serum from each group testedto determine the units of interferon present as described above. Theresults of these tests are tabulated below:

Divinyl ether-maleic anhydride Units of Interferon molecular weightPresent EXAMPLE 3 This example illustrates the effectiveness of adivinyl ethermaleic anhydride copolymer containing the monomers in a 1:2mole ratio and having an RSV of approximately 0.32 on guinea pigsinfected with foot-and-mouth disease virus.

Groups of eight guinea pigs, having an average weight of approximately500 g. each, were injected intraperitoneally with 25 mg. per guinea pigof divinyl ether-maleic anhydride copolymer in 0.5 ml. of 0.9 percentsterile saline solution. An equal number of control guinea pigs (averageweight 500 g. each) were injected intraperitoneally with 0.5 ml. of 0.9percent sterile saline solution. After l8 hours all the guinea pigs werechallenged with 0.1 ml. of various strength serial dilutions of type (O,BFS 1860 strain) foot-and-mouth disease virus given intradermallyto thepad of the right hind foot. From approximately '48 hours after thechallenge with footand-mouth disease virus the guinea pigs were examinedfor appearance of lesions at the primary site of challenge and at thesecondary sites (i.e., other feet and mouth). An arbitrary scale ofpossible lesions was set up and the percentage of lesions observed wascalculated. The results are tabulated below:

Percentage of Positive Sites After Challenge with ID. ID,.,' 1D,,copolymer treated guinea pigs 77.5 30 12.5 5.7

Control guinea pigs The term ID, stands for an infectious dose to 50percent of the animals being tested. Thus 4 ID is four times the dosewhich would be infectious to 50 percent of the animals.

EXAMPLE 4 Example 3 was repeated except the guinea pigs were challengedwith the foot-and-mouth disease virus either before treatment withdivinyl ether-maleic anhydride copolymer or simultaneously with thetreatment. The results are tabulated below: 1

Percentage of Positive Sites After Challenge with This exampleillustrates the effectiveness of a divinyl ethermaleic anhydridecopolymer containing the monomers in a 1:2 mole ratio and having an RSVof approximately 0.32 on suckling mice infected with foot-and-mouthdisease virus.

A stock solution of 20 milligrams per milliliter of the copolymer insterile saline solution was adjusted to a pH of 7.2 with 6N NaOl-l.Groups of IO suckling mice were injected intraperitoneally withdilutions of the above described copolymer solution and at various timeintervals thereafter, were challenged with I00 times the median lethaldose (LD of Asia l type foot-and-mouth disease virus. Controls weretreated exactly the same way except they were injected with salinesolution in place of the copolymer solution. Ten days after viruschallenge, the number of surviving mice were recorded. The amount ofcopolymer with which each suckling mouse was treated, the number of daysafter treatment the mice were challenged with foot-and-mouth diseasevirus, and the number of mice surviving (l0 days after challenge), aretabulated below:

Number 0! mice alive 10 days after virus challenge Concentration ofcopolymer 1 l Mlcrog'rams per 5 gm. mouse.

EXAMPLE 6 This example illustrates the effectiveness of divinylethermaleic anhydride copolymer on the seven major types offootand-mouth disease virus.

Following the procedure of example 5, groups of 20 suckling mice wereinjected intraperitoneally with 600 micrograms per 5 gm. mouse with theydivinyl ether-maleic anhydride copolymer solution described in example 5and 48 hours thereafter each group was challenged with 100 times themedian lethal dose of a different type foot-and-mouth disease virus.Seven days after virus challenge, the number of surviving mice wasrecorded. Groups of 10 suckling mice were used as controls and treatedexactly the same way except they were injected intraperitoneally withsaline solution in place of the copolymer solution. The types offoot-and-mouth disease virus used to challenge the mice and the numberof mice alive 7 days after the challenge (expressed a fraction of thetotal treated) are tabulated below:

Type of Foot-and-Mouth 7th Day Survival A 5/20 0/10 C 10/20 0/10 0 l5/200/10 What 1 claim and desire to protect by Letters Patent is:

l. A process of suppressing pathogenic foot-and-mouth disease virusgrowth which comprises administering to an animal infected with saidvirus an effective dosage of an antiviral agent selected from the groupconsisting of divinyl ether-maleic anhydride copolymers andphysiologically tolerated salts of divinyl ether-maleic anhydridecopolymers, said copolymers containing divinyl ether and maleicanhydride in a mole ratio of about l:2 and having a reduced specificviscosity of from about 0.04 to about 1.8.

2. A process of suppressing pathogenic foot-and-mouth disease viruswhich comprises inoculating eleven-footed animals with at least onedosage of from about 0.5 to 300 mgJkg. of body weight of an antiviralagent selected from divinyl ethermaleic anhydride copolymers andphysiologically tolerated salts of divinyl ether-maleic anhydridecopolymers, said copolymers containing divinyl ether and maleicanhydride in a mole ratio of about 1:2 and having a reduced specificviscosity of from about 0.04 to about l.8.

PO-l 050 V (5/69) UNITED STATES PATENT OFFICE CERTIFICATE OF CGRRECTIONPatent No. 3, 624, 218

Dated November 30, 1971- Inventofls) William Regelson (Case 1-1-4) It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

Column 4, In the Table for Example 3,

I ll II should appear under 4000 first column.

Signed and sealed this 25th day of April 1972.

EDWARD E LFLETCHER,JR,

0 ROBERT GOTTSCHALK Amer, 5mg Officer Cornmissinner' of Patents

2. A process of suppressing pathogenic foot-and-mouth disease viruswhich comprises inoculating cloven-footed animals with at least onedosage of from about 0.5 to 300 mg./kg. of body weight of an antiviralagent selected from divinyl ether-maleic anhydride copolymers andphysiologically tolerated salts of divinyl ether-maleic anhydridecopolymers, said copolymers containing divinyl ether and maleicanhydride in a mole ratio of about 1:2 and having a reduced specificviscosity of from about 0.04 to about 1.8.